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Biomedical subjects

F Pittner

Publications and source records attributed to F Pittner.

At least 19 recordsLinked to original sources
PubMed2005

Resonant nano-cluster devices.

The resonance-enhanced absorption (REA) by metal clusters on a surface is an effective technique on which to base bio-optical devices. A four-layer device consisting of a metal mirror, a polymer or glass-type distance layer, a biomolecule interaction layer and a sub-monolayer of biorecognitively bound metal nano-clusters is reported. Experiments indicate a strong influence of the resonator homogeneity on the absorption maximum. Layer stability plays an important role in the overall performance of the device. Techniques and optimised lab protocols to set up biochips that use the REA process in the detection are presented. The sensors show one to three narrow reflection minima in the visible and or infra-red (IR) part of the spectrum and therefore they do not suffer from the spectral limitations associated with spherical gold colloids. Metal clusters (synthesised by thermal step reduction) as well as metal- dielectric shell clusters (synthesised by various shell deposition processes) are used to precisely shift the readout of the device to any frequency in the visible and near IR range. Disposable single-step protein chips, DNA assays as well as complex biochip arrays are established that use various DNARNA, antigen-antibody and protein-protein interaction systems.

Biosensing Techniques↗
PubMed2003

Stabilisation and determination of the biological activity of L-asparaginase in poly(D,L-lactide-co-glycolide) nanospheres.

The preservation of biological activity of protein drugs in formulations is still a major challenge for successful drug delivery. The enzyme L-asparaginase, which exhibits a short in vivo half-life and is only active against leukaemia in its tetrameric form, was encapsulated in poly(D,L-lactide-co-glycolide) nanospheres by the (w/o)/w-emulsion solvent evaporation technique in presence of various potential stabilisers. Elucidation of the preparation steps revealed that the enzyme is denaturated at the aqueous/organic interface and by sonication. The preparation of L-asparaginase nanospheres with trehalose, PEG 400, and glycerol as components of the inner aqueous phase yielded colloidal formulations with increased biological activity as determined by an improved protocol for quantification of the active enzyme encapsulated. After lyophilisation the enzyme activity and particle size distribution were retained only by use of Pluronic F68 as a lyoprotectant. Despite the unaltered particle size and improved biological activity, the release profile of the enzyme was strongly altered by coencapsulation of the stabilisers resulting in increased first bursts. In consequence, the biological activity of L-asparaginase during preparation and storage can be improved by combining appropriate additives but concurrently the release profile is influenced.

Antineoplastic Agents↗
PubMed2001

High-throughput assays on the chip based on metal nano-cluster resonance transducers.

High throughput transducers using metal cluster resonance technology are based on surface-enhancement of metal cluster light absorption. These devices can be used for detection of biorecognitive binding, as well as structural changes of nucleic acids, proteins or any other polymer. The optical property for the analytical application of metal cluster films is the so-called anomalous absorption. An absorbing film of clusters positioned 10--400 nm to a mirror surface reacts in a similar way to a reflection filter. At a certain distance of the absorbing layer to the mirror the reflected electromagnetic field has the same phase at the position of the absorbing cluster as the incident fields. This feedback mechanism strongly enhances the effective cluster absorption coefficient. The system is characterised by a narrow reflection minimum whose spectral position shifts sensitively with the interlayer thickness, because a given cluster-mirror distance and wavelength defines the optimum phase. Based on this principle a set of novel tools including biochips and micro arrays is presented, which enabled us to transduce binding, as well as changes of protein-, DNA- and polymer-conformation, quantitatively into an optical signal which can be observed directly as a colour change of a sensor-chip surface.

Electromagnetic Fields↗
PubMed1999

Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media.

We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.

Animals↗
PubMed1997

Immobilization of active facilitated glucose transporters (GLUT-1) in supported biological membranes.

Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interference from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.

Biotin↗
PubMed1996

Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.

A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.

Amino Acid Sequence↗
PubMed1996

Determination of salicylate in beverages and cosmetics by use of an amperometric biosensor.

A fast and selective enzymatic method for the determination of salicylate in beverages and cosmetics has been developed. The enzyme salicylate hydroxylase was immobilised covalently onto a glassy carbon working electrode of a wall-jet cell coupled with a flow-injection analysis system. The salicylate is enzymatically converted to catechol, which can be detected amperometrically on the glassy carbon electrode at +0.45 V. The response of the biosensor is linearly proportional to the concentration of salicylate between 725 nmol/l and 700 micromol/l. A high sample throughput (60 h(-1)) is possible, and the biosensor is stable for more than three months. Sample pretreatment for beverages and hair lotions is easy and fast. For creams, an extraction of salicylate is necessary. Relative standard deviations are less than 5.5% and the recoveries are between 95 and 105%.

Journal Article↗
PubMed1995

Drug-protein conjugates: preparation of triamcinolone-acetonide containing bovine serum albumin/keyhole limpet hemocyanin-conjugates and polyclonal antibodies.

A radioimmunoassay has been developed for the quantitation of triamcinolone-acetonide (TAAc) at the picogram level. For use of TAAc as an antigenic epitope first the drug was hemisuccinoylated at C-21 as confirmed by 13C-NMR- and mass spectroscopy after derivatization. This hapten was conjugated to the carrier-protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) by different amide-bond generating methods (imidazolide-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, mixed anhydride-method) yielding antigens of quite different conjugation number, solubility and usefulness. The mixed anhydride-method yielded most useful soluble conjugates bearing 0.3-31.5 mol TAAc per mol carrier-protein. Coupling by the carbodiimide-method yielded insoluble conjugates, inappropriate for antigen synthesis in hapten immunoassays because of formation of coupling agent modified residues and crosslinking of the carrier-protein. Specificity of the antisera obtained by immunization with TAAc-BSA and TAAc-KLH was assessed by isolation of the soluble hapten-antibody complex and a RIA protocol was developed providing a detection limit of 200 pg (0.46 pmol) TAAc/ml sample.

Antibodies, Monoclonal↗
PubMed1995

Drug-protein conjugates: haptenation of 1-methyl-10 alpha-methoxydihydrolysergol and 5-bromonicotinic acid to albumin for the production of epitope-specific monoclonal antibodies against nicergoline.

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5-bromonicotinic acid and 1-methyl-10 alpha-methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond-generating methods (mixed anhydride-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, and dicyclohexylcarbodiimide/N-hydroxysuccinimide methods) were used in bovine serum albumin (BSA)-coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23 mol of 1-methyl-10 alpha-methoxydihydrolysergol (MMD) or 41 mol of 5-bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten-inhibition ELISA using antigen-coated wells indicate that the supernatant of MMD-specific hybridoma exhibited 50% inhibition of antibody binding at 17 +/- 2 micrograms of MMD and at 24.5 +/- 2 micrograms of nicergoline, and the BNA-specific hybridoma exhibited similar inhibition at 147 +/- 6 micrograms of BNA and 500 +/- 30 micrograms of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten-inhibition ELISA.

Alkaloids↗
PubMed1992

Construction and use of two alpha-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype alpha-hANP biosensor.

Two alpha-human atrial natriuretic peptide (alpha-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of alpha-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate alpha-hANP-specific antibodies from a goat anti-alpha-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an alpha-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological alpha-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This alpha-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.

Amino Acid Sequence↗
PubMed1990

Immobilization of alpha-human atrial natriuretic peptide on insoluble supports and affinity purification of specific antibodies from a polyclonal goat anti-alpha-human atrial natriuretic peptide serum.

alpha-Human atrial natriuretic peptide (alpha-hANP) was covalently coupled via single attachment onto two different insoluble matrices. Controlled-pore glass-alpha-hANP matrices were well suited for the purification of monospecific antibodies, whereas Enzacryl AA-alpha-hANP did not withstand the inevitable chemical and physical stresses during affinity purification.

Animals↗
PubMed1990

Determination of alpha-human atrial natriuretic peptide (alpha-hANP) in urine using combination HPLC with RIA strongly indicates non-immunoreactive metabolites.

Employing HPLC coupled with RIA, it was shown that alpha-human atrial natriuretic peptide is excreted in urine. Freshly collected urine had to be acidified to obtain reproducible results. When prepurified urine was subjected to HPLC (ion exchange and reversed phase) the subsequent quantification of alpha-hANP immunoreactive material in the eluate showed 10- to 30-fold greater amounts of alpha-hANP after treatment with HPLC; substances with the same elution parameters as synthetic alpha-hANP were detected, but they gave no response in the RIA.

Atrial Natriuretic Factor↗
PubMed1988

Studies on the immobilization of glucuronidase (Part 2). Cleavage of hardly soluble substrates in organic solvents.

Naturally occurring glucuronides and glucosides dissolved in organic solvents can be split with the help of beta-glucuronidase (EC 3.2.1.31) immobilized on controlled pore glass. To protect the enzyme against denaturation by the organic solvents and to promote hydrolytic cleavage of substrates, two methods were used: (a) Immobilization via crosslinking with aged glutaraldehyde in presence of bovine serum albumin; and (b) Adsorption of wet enzyme to the carrier in the presence of organic solvents.

Cross-Linking Reagents↗
PubMed1988

Degradation of bacterial cell walls by immobilized lysozyme.

Lysozyme was immobilized by two different methods in two different ways in order to obtain a preparation with an activity as high as possible toward a macromolecular substrate. The enzyme was bound as a Schiff base to a silicate carrier by using oxidized dextrans of different lengths as spacer and also was bound to controlled pore aminoglass via pyridino-4-aldehyde and BrCN. The latter preparations had activities up to 2.5% of the free lysozyme.

Buffers↗